The development of new drugs and biologically active substances for therapeutic, diagnostic and research reagent purposes traditionally concentrates on the rational design of such compositions based upon knowledge of the target biomolecule, i.e. the molecule to be modulated.
Bodily states in mammals, including many disease states, are effected by biomolecules. Classical therapeutics has generally focused upon interactions with proteins in efforts to modulate disease-potentiating functions of such proteins. In addition to modulating disease-potentiating functions of proteins directly, recent attempts have been made to moderate the actual production of proteins by interactions with molecules that direct their synthesis, intracellular RNA. Biological functions may also be modulated or regulated by interactions with other biomolecules such as nucleic acids, carbohydrates, lipids, steroids or toxins.
One approach for constructing therapeutics, diagnostics and research reagents has been simple modifications of known amino acid or nucleic acid sequences. Such techniques are limiting because the number of individual sequences of simple modifications necessary for the development of new substances is prohibitively large. In addition, many drug targets and other target molecules are too extensive and complex to be analyzed by these mutational experiments. Other biomolecule targets, by virtue of their particular chemical nature, are not candidates for directed mutagenesis of this sort. Some examples of such biomolecules are carbohydrates, lipids, and steroids.
Recently, methods have been devised whereby therapeutics, diagnostics and research reagents can be developed more quickly. A variety of combinatorial strategies have been described to identify active peptides. Houghton, et al. Nature 1991, 354, 84; Lam, et al., Nature 1991, 354, 82; Owens, et al., Biochem. Biophys. Res. Commun. 1991, 181, 402; Fodor, et al., Science 1991, 251, 767; Geysen, et al., Molecular Immunology 1986, 23, 709; Zuckermann, et al., Proc. Natl. Acad. Sci. 1992, 89, 4505; Rutter, et al., U.S. Pat. No. 5,010,175 issued Apr. 23, 1991; Lam, et al., PCT US91/06444 filed Jul. 1, 1991, Dooley, et al., Proc. Natl. Acad. Sci. U.S.A., 1993, 90, 10811; Dooley and Houghten, Life Sciences, 1993, 52, 1509; Ohlmeyer, et al., Proc. Natl. Acad. Sci. U.S.A., 1993, 90, 10922; Jayawickreme, et al., Proc. Natl. Acad. Sci. U.S.A., 1994, 91, 1614.
Rutter, et al. describes a method whereby statistically randomized peptides may be prepared and active peptide selected for and identified.
Lam, et al., teaches the preparation of randomer libraries, especially random peptide libraries in which each randomer sequence is individually coupled to a solid support (i.e. one oligomer sequence/one bead). Further, a reporter group is attached to each oligomer/bead in order to identify active oligomers from inactive oligomers. The randomer libraries of Lam, et al., PCT US91/06444 filed Jul. 1, 1991, are contacted with a target biomolecule and active randomers, identified via reporter groups, are isolated using the solid support to manually remove the active randomer from the rest of the library. Lam, et al. further teaches that the selected randomer can be characterized such as by Edman degradation or FAB-MS.
Combinatorial strategies for nucleic acids have also been developed. Such methods generally select for a specific nucleic acid sequence from a pool of random nucleic acid sequences based on the ability of the selected sequence to bind to a target protein. The selected sequences are then commonly amplified and the selection process repeated until a few strongly binding sequences are identified. Commonly, the pool of random nucleic acid sequences is comprised of short random sequences embedded in external flanking or "carrier" nucleic acid molecules of known sequence. Such carrier molecules are intended to neither enhance, nor detract from binding of the oligonucleotide. The amplification primers are prepared to be complementary to known sequences of the "carrier" generally of lengths 20 nucleotides in length or more. Such "carrier" portions are meant to facilitate manipulation of the molecule and preferably have neutral effect upon the randomized sequence to be selected for. Using this method Tuerk and Gold, Science 1990, 249,505; identified a sequence which strongly binds T4 polymerase binding protein, gp43, but which would not have been predictable using traditional methods. Ellington and Szostak, Nature 1990, 346, 818; identified sequences which bind small ligands using this method, Bock, et al., Nature 1992, 355, 564; designed DNA molecules which recognize the protease thrombin and Schneider, et al., J. Mol. Biol., 1992, 228, 862; isolated RNA ligands with high affinity for the bacteriophage R17 coat protein. More recently, using this method, Schneider, et al., FASEB J., 1993, 7, 201; identified RNA molecules from a pool of RNA molecules that bind tightly to the E. coli transcription termination factor rho and Jellinek, et al., Proc. Natl. Acad. Sci. U.S.A., 1993, 90, 11227; isolated RNA ligands with low-nanomolar affinity and high specificity to basic fibroblast growth factor from a library of RNA molecules.
Randomer oligonucleotide libraries are also mentioned by Lam, et al. Characterization of selected oligonucleotides after manual separation is suggested using the techniques of Maxam and Gilbert or by the use of an oligonucleotide sequencer. Electrospray-high performance mass spectrometry is also suggested in order to determine sequences and structures of randomers. These methods require the addition of a reporter group to the oligomer species in order to identify and manually isolate active oligomers. Furthermore, randomer libraries are limited to oligomers approximately 5 mers in length since preparation of more than 5 mers would present unwieldy amounts of oligomer/beads as well as the need for large amounts of target.
While some advantages have been achieved by the foregoing methods, simple methods of determining therapeutics, diagnostics, and research reagents are desirable. Previous methods have relied upon complex nucleic acid molecules comprised of at least partially random sequence portions to be selected for, as well as lengthy flanking carrier portions necessary to support the reactions. Still other methods are limited in the length of oligonucleotides within the randomer library which is physically possible. Methods requiring enzymatic amplification of active sequences are often limited by incompatibility with many non-standard nucleotides or nucleosides.
Methods in which selection may be carried out with a fully sequence-randomized pool of discrete molecules and a minimum of other fixed sequences would be greatly desirable as they would increase specificity, facilitate manipulation of the pool of molecules, and eliminate any indeterminate bias in binding selection resulting from the presence of either internal sequence fixed (unrandomized) positions and/or the carrier portions.
Such improved methods to determine oligonucleotides for antisense therapeutics, diagnostics and research reagents are greatly desired.
Although oligonucleotides are currently being administered as therapeutic agents, it is not known a priori how to select an ideal nucleotide sequence to bind selectively to a target molecule. Moreover, in many cases, it is even difficult to decide what region of a gene or protein, for example, to target in order to achieve maximum effect. The methods of the present invention overcome these difficulties.